The present invention provides antibodies against human antigens and methods for generating high affinity antibodies against these targets. More particularly, the invention provides humanized or human antibodies against vascular endothelial growth factor (VEGF) and methods for generating such anti-VEGF antibodies. In addition, the invention provides compositions, kits and methods of using these antibodies and derivatives thereof to inhibit angiogenesis in vitro, and for diagnosing or treating diseases associated with abnormal angiogenesis such as cancer, rheumatoid arthritis, ischemic-reperfusion related brain edema and injury, cortical ischemia, ovarian hyperplasia and hypervascularity, endometriosis, psoriasis, diabetic retinopaphy, and other ocular angiogenic diseases.
Angiogenesis has been involved in many physiological and pathological processes. Angiogenesis consists of multiple steps that ultimately resulting in proliferation and differentiation of endothelial cells, and formation of tubes and cavities (angiogenesis). Factors that promote angiogenesis include VEGF, aFGF, bFGF, TGF-α, TGF-β, HGF, TNF-α, angiogenin, IL-8, etc., whereas factors that inhibit angiogenesis include thrombospondin (Good et al. Proc. Natl. Acad. Sci. USA. 87:6624-6628 (1990)), the N-terminal fragment of prolactin (Clapp et al. Endocrinology, 133:1292-1299 (1993)), kringle 5 domain of plasminogen (Cao et al., J. Biol. Chem., 271:29461-29467 (1996), angiostatin (O'Reilly et al. Cell, 79:315-328 (1994)) and endostatin (O'Reilly et al. Cell, 88:277-285 (1996)).
Vascular endothelial cell growth factor (VEGF) is a growth factor acting specifically with its receptors on vascular endothelial cells to promote their angiogenesis. It is known that angiogenesis plays an important role in the development of new vasculature from preexisting blood vessels and/or circulating endothelial stem cells (Asahara et al., Science, 275(5302):964-967, 1997; Springer et al., Mol. Cell, 2(5):549-558, 1998; Folkman and Shing, J. Biol. Chem., 267:10931-10934,1992). Angiogenesis also plays a vital role in many physiological processes, such as embryogenesis, wound healing and menstruation. More importantly, angiogenesis is further involved in pathological conditions such as tumor formation, metastasis, diabetic retinopathy, etc. It is known that the growth of a solid tumor requires tumor vascularization for supplying oxygen and nutrients and the metastasis of tumor cells occurs through blood vessels resulting from the tumor vascularization. VEGF is believed to be a pivotal angiogenic factor in this vascularization for tumors. Therefore, it is expected that the growth and metastasis of tumor can be inhibited by certain substances neutralizing the vascularization activity of VEGF. Recent studies (Burrows and Thorpe, Pharmacol. Ther., 64:155-174, 1994; Proc. Natl. Acad. Sci. USA, 90:8996-9000, 1994) have used such a strategy to target the vasculature of solid tumors. Targeting the blood vessels of the tumors, rather than the tumor cells themselves, has certain advantages in that it is not likely to lead to the development of resistant tumor cells, and that the targeted cells are readily accessible. Moreover, destruction of the blood vessels leads to an amplification of the anti-tumor effect, as many tumor cells rely on a single vessel for their oxygen and nutrients
In nearly half of diabetics diabetic retinopathy occurs as one of complications of diabetes. It is believed that the formation of microcapillaries is promoted in diabetic retinopathy by oxygen deficiency. These microcapillaries will sooner or later be ruptured to bleed to form scar tissue, leading to detached retinas. Age-related macular degeneration is another eye disease that has been demonstrated to be involved in pathological vascularization in the retina. Hence, it is expected that inhibition of vascularization can prevent retinopathy from developing. Based on experiments using monkey, Miller et al. reported that VEGF is related very closely to the development of vegetative retinopathy (Miller et al.: Am. J. Pathol. 145, 574-584 (1994)). For this reason, a substance neutralizing the vascularization activity of VEGF is considered useful for preventing or treating diabetic retinopathy and AMD (Lopez et al. Invest. Ophtalmo. Vis. Sci. 37:855-868 (1996)).
The recognition of VEGF as a primary stimulus of angiogenesis in pathological conditions has led to various attempts to block VEGF activity. Inhibitory anti-VEGF receptor antibodies, soluble receptor constructs, antisense strategies, RNA aptamers against VEGF and low molecular weight VEGF receptor tyrosine kinase (RTK) inhibitors have all been proposed for use in interfering with VEGF signaling (Siemeister et al., Cancer Metastasis Rev., 17(2):241-248., 1998). In fact, monoclonal antibodies against VEGF have been shown to inhibit human tumor xenograft growth and ascites formation in mice (Kim et al., Growth Factors 7:53 (1992); Nature 362:841-844 (1993); Asano et al., Hybridoma, 17:185-90, (1998); Mesiano et al., Am. J. Pathol., 153(4):1249-1256, (1998); Luo et al., Cancer Res., 58(12):2594-2600, (1998); Cancer Res., 58(12):2652-2660, (1998); Borgstrom et al. Cancer Res. 56:4032-4039 (1996); Borgstrom et al., Prostate, 35(1): 1-10, (1998)). Furthermore, the same strategy using anti-angiogenic molecules, anti-VEGF antibody, and VEGF antagonists have been utilized in experimental treatment of AMD and diabetic retinopathy (Adamis et al. Arch. Ophthalmol. 114:66-71 (1996)). For the therapeutic applications, antibodies are generally engineered to reduce their toxicities in repeated dosage by humanization, if they are derived originally from mouse and to improve other attributes such as binding affinity with the target molecules by affinity maturation (Winter and Milstein, Nature, 349:293-299, (1991); Baca et al, J. Biol. Chem., 272(16):10678-84, (1997); Presta, et al., Cancer Res., 57:4593-4599, (1997); Chen et al. (1999) J. Mol. Biol. 293:865-881; and Ryan et al. (1999) Toxicologic Pathology, 27(1):78-86).
Although the foregoing studies underscore the importance of VEGF in solid tumor growth, and its potential as a target for tumor therapy, the identification of additional agents that inhibit VEGF-induced angiogenesis would be of benefit in expanding the number of therapeutic options. The development of therapeutic agents that specifically inhibit VEGF to bind with its receptor represents important alternatives to target angiogenesis more effective with potentially improved therapeutic benefits.